The invention relates to a method for enzymatically synthesising an (oligo)peptide (i.e. a peptide, in particular oligopeptide), to an enzyme suitable for catalyzing said synthesis, to a host cell capable of functionally expressing said enzyme and to a method for preparing said enzyme.
Peptides, in particular oligopeptides have many applications, for instance as pharmaceutical, food or feed ingredient, or cosmetic ingredient.
Methods for synthesizing (oligo)peptides are generally known in the art.
Oligopeptides can be chemically synthesized in a stepwise fashion in solution or on the solid phase via highly optimized processes. However, peptides longer than 10-15 amino acids are often very difficult to synthesize due to side reactions and as a consequence purification is troublesome. Therefore, peptides longer than 10 amino acids are often synthesized by a combination of solid-phase synthesis of side-chain protected oligopeptide fragments which are subsequently chemically condensed in solution, e.g. as in a 10+10 condensation to make a oligopeptide of 20 amino acids. The major drawback of chemical side-chain protected oligopeptide fragment condensation is that upon activation of the C-terminal amino acid residue of the acyl donor racemisation occurs. In contrast, enzyme-catalyzed peptide couplings are completely devoid of racemisation and have several other advantages over chemical peptide synthesis such as the absence of side reactions on the side-chain functionalities. For industrial application, an enzymatic peptide synthesis concept based on a kinetic approach, i.e. using an acyl donor C-terminal ester is most attractive (see for instance N. Sewald and H.-D. Jakubke, in: “Peptides: Chemistry and Biology”, 1st reprint, Ed. Wiley-VCH Verlag GmbH, Weinheim 2002).
Chemo-enzymatic peptide synthesis can entail the enzymatic coupling of oligopeptide fragments which have individually been synthesized using chemical synthesis, fermentation, or by a combination of chemical and enzymatic coupling steps. Some reports have been published on the enzymatic condensation of oligopeptide fragments in aqueous solution (Kumaran et al. Protein Science, 2000, 9, 734; Bjorup et al. Bioorg. Med. Chem. 1998, 6, 891; Homandberg et al. Biochemistry, 1981, 21, 3387; Komoriya et al. Int. J. Pep. Prot. Res. 1980, 16, 433). However, a major drawback of such enzymatic oligopeptide fragment condensation in aqueous solution is that simultaneous hydrolysis of the peptide bonds within the oligopeptide fragments and of the C-terminal ester functionality takes place leading to low yields and many side products.
Proteases have hitherto mainly been produced commercially for hydrolytic application, e.g. in cleaning, where peptide bonds are hydrolysed by the proteases. A typical example are the subtilisins, which form an enzyme class with considerable importance for their use as detergents. Therefore, subtilisins have been the subject of numerous protein engineering studies. subtilisins have also been used for the synthesis of oligopeptides, which was, however, almost always accompanied by hydrolytic side-reactions to a significant extent. It was found by Wells et al. (U.S. Pat. No. 5,403,737) that the condensation of oligopeptides in aqueous solution could be significantly improved by altering the active site of subtilisin BPN′, a subtilisin from B. amyloliquefaciens (SEQUENCE ID NO: 2). When two mutations were introduced, i.e. S221C and P225A, a subtilisin BPN′ variant called subtiligase was obtained having a 500-fold increased synthesis over hydrolysis ratio (S/H ratio) as compared to wild-type subtilisin BPN′. However, the average ligating yield was around 66% and hydrolysis of the oligopeptide acyl donor C-terminal ester was still substantial (Wells et al. Science, 1994, 266, 243). Most often, 10 equivalents of oligopeptide acyl donor C-terminal ester was used to obtain a decent reaction yield. Another drawback of subtiligase was the poor stability against organic co-solvents that are required to solubilize the oligopeptide fragments, against enhanced temperature and against denaturating agents, which are often needed for successful oligopeptide condensation. Therefore, Wells et al. added five additional mutations to subtiligase, i.e. M50F, N76D, N109S, K213R and N218S, to make the enzyme more stable (Proc. Natl. Acad. Sci. USA, 1994, 91, 12544). The new mutant called stabiligase appeared moderately more resistant to sodium dodecasulfate and guanidinium hydrochloride, but hydrolysis was still a major side reaction. For instance an oligopeptide carboxyamidomethyl-ester (Cam-ester) was ligated to an oligopeptide amine using stabiligase in a yield of 44%. In this example, 10 equivalents of the oligopeptide C-terminal ester were used and thus, 9.56 equivalents of the oligopeptide C-terminal ester were hydrolyzed at the C-terminal ester functionality and only 0.44 equivalents ligated to the oligopeptide amine to form the product. Clearly, there is a need for an improved enzyme with a higher S/H ratio to make the oligopeptide condensation reaction an economically viable process. Probably for this reason, the past 20 years subtiligase nor stabiligase have been industrially applied, to the best of the inventors knowledge.
Another aspect of subtilisin BPN′ that has received attention is the increase of the stability of the enzyme for its use as detergent (i.e. for the hydrolysis of peptide bonds) at higher temperatures and/or in the presence of metal chelators. A typical example of such study was disclosed by Bryan et al. who engineered a subtilisin BPN′ variant lacking a high affinity Ca2+ binding site (WO02/22796). The high affinity Ca2+ binding site in subtilisin BPN′ is made up by a loop comprising amino acids 74-82 and the amino acids Gln2 (Q2) and Asp41 (D41). Comparison of the 3D structure of subtilisin BPN′ with the structure of homologous subtilisins shows that the high affinity Ca2+ binding site is highly conserved. This binding site is important for their stability in known subtilisins. Stripping of the Ca2+ ion by for instance metal chelators leads to unfolding and thus inactivation of the known subtilisins. When Bryan et al. deleted amino acids 75-83 (Δ 75-83) of subtilisin BPN′ and additionally implemented the mutations Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, Y217L, N218S, T254A and Q271E, a subtilisin BPN′ variant was obtained (called BS149, also known as Sbt149) which lacks the Ca2+ binding domain and has a greatly improved stability against metal chelators (1000×). However, this enzyme cannot be used for peptide fragment condensation in aqueous solution since it is only hydrolytically active.
It is an object of the present invention to provide an enzymatic method for preparing an (oligo)peptide by condensation of a first and a second (oligo)peptide fragment or by cyclisation of an (oligo)peptide that can serve as an alternative to known methods of preparing (oligo)peptides. There is a need for alternative methods in general, in particular in order to broaden the palette of tools for making specific (oligo)peptides.
In particular, it is an object to provide an enzymatic method for preparing an (oligo)peptide by condensation of a first and a second (oligo)peptide fragment or by cyclisation of an (oligo)peptide, wherein an enzyme is used having an improved S/H ratio and stability compared to subtilisin BPN′, at least under certain reaction conditions.
Further, it is an object to provide an enzymatic method for preparing an (oligo)peptide by condensation of a first and a second (oligo)peptide fragment or by cyclisation of an (oligo)peptide, wherein an enzyme is used having an improved stability compared to subtiligase, in particular an improved stability and S/H ratio compared to subtiligase.
It is yet a further object of the invention to provide the coupling of a (oligo)peptide to a protein. It is in particular a challenge to provide enzymatic methodology that allows coupling of a peptide with a protein, in particular due to the added complexity of a protein's three-dimensional structure.
It is yet a further object to provide a novel subtilisin BPN′ variant capable of catalyzing the condensation of two (oligo)peptides or of cyclisation of an (oligo)peptide, in particular such an enzyme having an improved property, such as an improved synthesis over hydrolysis ratio ratio and/or improved stability, compared to known enzymes suitable to catalyse such condensation, such as subtilisin BPN′ and/or subtiligase, at least under certain reaction conditions.
One or more other objects that may be subject of the invention follow from the description below.
It has now surprisingly been found that it is possible to provide a subtilisin BPN′ variant wherein the calcium binding domain at the positions corresponding to amino acids 75-83 has been inactivated, namely by deletion, that has catalytic activity with respect to the condensation of two (oligo)peptide fragments or the cyclisation of a peptide, and in particular to provide such a variant that has an improved S/H ratio compared to subtilisin BPN′ and/or subtiligase, by providing a subtilisin BPN′ variant that has a specific mutation, preferably a specific combination of mutations, in addition to the deletion of the amino acids corresponding to positions 75 to 83.
Accordingly, the present invention relates to a method for enzymatically synthesizing an (oligo)peptide, comprising coupling (a) an (oligo)peptide C-terminal ester or thioester and (b) an (oligo)peptide nucleophile having an N-terminally unprotected amine,
wherein the coupling is carried out in a fluid comprising water, and
wherein the coupling is catalyzed by a subtilisin BPN′ variant or a homologue thereof, which comprises the following mutations compared to subtilisin BPN′ represented by SEQUENCE ID NO: 2 or a homologue sequence thereof:
i) a deletion of the amino acids corresponding to positions 75-83;
ii) a mutation at the amino acid position corresponding to S221, the mutation being S221C or S221 selenocysteine (S221U);
iii) preferably, a mutation at the amino acid position corresponding to P225;
wherein the amino acid positions are defined according to the sequence of subtilisin BPN′ represented by SEQUENCE ID NO: 2.
Further, the invention relates to a method for enzymatically synthesizing a cyclic (oligo)peptide of at least 12 amino acids, comprising subjecting an (oligo)peptide C-terminal ester or thioester having an N-terminally unprotected amine to a cyclisation step wherein said cyclization is carried out in a fluid comprising water, and
wherein the cyclization is catalyzed by a subtilisin BPN′ variant or a homologue thereof, which comprises the following mutations compared to subtilisin BPN′ represented by SEQUENCE ID NO: 2 or a homologue sequence thereof:
i) a deletion of the amino acids corresponding to positions 75-83;
ii) a mutation at the amino acid position corresponding to S221, the mutation being S221C or S221 selenocysteine;
iii) preferably, a mutation at the amino acid position corresponding to P225;
wherein the amino acid positions are defined according to the sequence of subtilisin BPN′ represented by SEQUENCE ID NO: 2.
Further, the invention relates to an enzyme, which enzyme is a subtilisin BPN′ variant or homologue thereof, comprising the following mutations compared to subtilisin BPN′ represented by SEQUENCE ID NO: 2 or homologue sequence thereof:
i) a deletion of the amino acids corresponding to positions 75-83;
ii) a mutation at the amino acid position corresponding to S221, the mutation being S221C or S221 selenocysteine;
ii) a mutation at the amino acid position corresponding to P225;
in which the amino acid positions are defined according to the sequence of subtilisin BPN′ represented by SEQUENCE ID NO: 2.
Further, the invention relates to a recombinant method for preparing the enzyme according to the invention, said method comprising:                a) providing a recombinant host cell functionally expressing a gene encoding the enzyme;        b) culturing said host cell under conditions which provide for the expression of the enzymatically active enzyme; and        c) recovering the expressed enzyme from said microbial host.        
Further, the invention relates to a recombinant polynucleotide comprising a sequence which encodes for an enzyme according to the invention.
Further, the invention relates to a host cell, comprising a polynucleotide according to the invention. The host cell is capable of functionally expressing the enzyme of the invention.
Further, the invention relates to the use of an enzyme according to the invention as a catalyst. Such use generally comprises contacting one or more substrates (reactants) in the presence of the enzyme under conditions wherein the enzyme catalyses a chemical reaction wherein the substrate(s) participate(s). The enzyme has been found particularly useful as a catalyst in peptide synthesis. It is in particular contemplated that an enzyme of the invention is useful for catalyzing reactions of which known subtilisins are known to be catalytically active. In an embodiment, the synthesised peptide is a protein. In an embodiment the synthesised peptide is an oligopeptide. In a further embodiment the synthesised peptide is composed of at least 201 amino acid units.
The invention provides a useful alternative to known methods of preparing (oligo)peptides, including proteins extended with an (oligo)peptide.
Moreover, it has surprisingly been found possible with a method of the invention to enzymatically condense two (oligo)peptide fragments or to cyclize an (oligo)peptide in a liquid comprising water with a high synthesis over hydrolysis ratio. The method of the invention is advantageous in that it offers the possibility for coupling various oligopeptide fragments in aqueous solution in high yield without substantial hydrolytic side reactions. Such surprising finding is illustrated by the Examples, which show that a method of the invention is not only suitable to synthesise (oligo)peptides that lack a secondary and tertiary protein structure, but also allows coupling two peptide fragments wherein at least one of the fragments is a protein, thereby synthesizing an (elongated) protein provided with an additional sequence of amino acid units. It has been found possible to synthesise such protein whilst maintaining a secondary and tertiary structure of the protein.
For the purpose of this invention, with “synthesis over hydrolysis ratio” (S/H ratio) is meant the amount of enzymatically synthesised (oligo)peptide product divided by the amount of (oligo)peptide C-terminal ester or thioester of which the ester or thioester group has been hydrolysed.
The value of the S/H ratio of an enzyme of the invention depends on various factors, for instance the nature of the substrates (the amino acid sequences of the (oligo)peptide C-terminal ester or thioester and of the (oligo)peptide nucleophile) and reaction conditions (e.g. temperature, pH, concentration of the peptide fragments, enzyme concentration). As shown in the Examples, it was found though that under various reaction conditions and for various substrates the S/H ratio was higher than for known subtilisins, such as subtiligase and subtilisin BPN′. Thus, it is contemplated that the S/H ratio of an enzyme according to the invention in general has a significantly higher S/H ratio than subtiligase and subtilisin BPN′, when tested under the same reaction conditions and using the same substrates, and in particular it is contemplated that an enzyme of the invention has a significantly higher S/H ratio under the conditions used in Example 1 (100 mM phosphate buffer, pH 8.0, temperature about 20° C., concentration of (oligo)peptide C-terminal ester 0.83 mM, concentration of (oligo)peptide nucleophile 3.33 mM, enzyme concentration 5.5 mg/L) or one or more of the other examples. Thus, in particular, the invention relates to a subtilisin BPN′ variant or homologue thereof wherein the S/H ratio of the subtilisin BPN′ variant or homologue thereof divided by the S/H ratio of subtiligase—at least under the conditions described in Example 1 or one or more of the other Examples—is more than 1, preferably 2 or more, in particular 5 or more. The upper value of this quotient is not critical; in practice it may e.g. be 100 or less, in particular 20 or less.
The S/H ratio of the subtilisin BPN′ variant or homologue thereof of the invention divided by the S/H ratio of subtilisin BPN′—at least under the conditions described in Example 1 or one or more of the other Examples—is usually more than 100, preferably 250 or more, more preferably 500 or more, in particular 1000 or more. The upper value of this quotient is not critical; The S/H ratio of subtilisin BPN′ at least under the reaction conditions specified herein is generally very low, it may be even zero (no detectable synthesis). Thus, the S/H ratio of the subtilisin BPN′ variant or homologue thereof of the invention divided by the S/H ratio of subtilisin BPN′ may approximate infinity. In a potential circumstance wherein subtilisin BPN′ has substantial ligase or cyclase activity, the inventors consider that the S/H ratio of the subtilisin BPN′ variant or homologue thereof of the invention divided by the S/H ratio of subtilisin BPN′ is also high, e.g. up to 100 000, in particular up to 25 000, more in particular up to 10 000.
Further, using a method of the invention, the (oligo)peptide product is very easy to purify from the reaction mixture because only little hydrolytic by-products are formed.
Another advantage of the invention is that, due to the improved S/H ratio, a small or no excess of the (oligo)peptide C-terminal ester or thioester or of the (oligo)peptide nucleophile is needed to reach a high yield (>80%) in the condensation reaction. Accordingly, in an advantageous embodiment an (oligo)peptide C-terminal ester or thioester and an (oligo)peptide nucleophile are contacted in a small excess of one of said (oligo)peptide fragments or in an about stoichiometric ratio although a larger excess of one over the other may be used, as described below.
As illustrated by the Examples, an enzyme according to the invention is also advantageous in that it allows the synthesis of a cyclic (oligo)peptide with significantly higher yield than with subtiligase (78% versus 61% for subtiligase). Cyclic (oligo)peptides are a particularly interesting class of peptides since they are often more potent due to their more constrained three dimensional structure and higher resistance to proteolysis.